The protective properties of the endotoxin protein

The isolation and properties of endotoxin protein, or lipid A-associated protein (LAP), from Shigella sonnei were described earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 11-17, and No. 7). In this report the data on its protective activity are presented. In experiments on mice one nanogram of LAP injected i. v. protected 50% of the animals against i. p. challenge with 40 LD50 of virulent S. sonnei. Guinea pigs injected s. c. with 10 micrograms of LAP were protected against local (keratoconjunctival) challenge with S. sonnei, the efficiency of immunization being 58%. LAP preparations containing no detectable amounts of O-antigen (less than 0.003%) were found to have a protective effect. Hyperimmune anti-LAP rabbit serum prevented local infection when incubated with S. sonnei challenge inoculum before injection into guinea pigs. Both active and passive protection induced by LAP was specific since no effect was observed in animals challenged with Shigella flexneri. In the homologous system the protective effect of anti-LAP serum was abolished by the addition of protein-free LPS. These results are compatible with the hypothesis that the protective activity of LAP depends on the presence of minute amounts of O-antigen whose immunogenic effect is greatly amplified by the protein component of the natural endotoxin complex.     

Experimental study of the preventive properties of immune milk containing Shigella antibodies

The protective properties of the mammary gland secretions of cows immunized with Shigella sonnei into the udder were studied. As a model for this study the intranasal and intraperitoneal infection of white mice was used. Immune milk was found to have pronounced protective properties against S. sonnei. When introduced intraperitoneally, this milk protected the animals infected with S. sonnei from death. When introduced intranasally, it not only protected the animals from death, but perceptibly inhibited the development of the pathological process in the pulmonary tissue, preventing the multiplication of shigellae and accelerating the elimination of the infective agents from the lungs of the infected animals. As a rule, the degree of protective action was determined by the level of antibodies to shigellae in the substrate under test.     

Immunogenic complexes of the soluble surface antigens of Sh. sonnei]

The comparative study of the immunogenic properties of Sh. sonnei (phases I and II) soluble surface antigens obtained by the modified method of aqueous-saline extraction and Sh. sonnei (phase I) antigen obtained by Boivin's method was made with the use of the keratoconjunctival test in guinea pigs. The protective activity of a high molecular fraction obtained by the fractionation of phase I soluble surface antigens in Sepharose 4B was studied. Boivin's antigen, when used for immunization in optimum doses, was found to have pronounced protective properties, whereas phase II soluble surface antigens showed no protective activity. A high molecular fraction obtained from phase I soluble surface antigen was found to be the most immunogenic. Protective activity was largely connected with protein antigen. The question whether protein antigen was an independent protective antigen or whether it constituted a part of a complex which determined the protective activity of a high molecular fraction remained unsolved.     

The effect of Shigella sonnei strains opposite in their virulence on different links in the immune system]

For the first time different action of S. sonnei strains, opposite in their virulence, on hematopoiesis and the functional activity of T- and B-lymphocytes has been shown. The hematopoiesis-disturbing action of virulent shigellae is manifested by their capacity, more pronounced than similar capacity of an avirulent (vaccine) strain, for stimulating the processes of endo- and exocolony formation, the proliferation of hematopoietic stem cells and their migration to the blood. The effect produced by shigellae on T-cell-mediated immune response is manifested by the suppression of macrophage migration and its subsequent activation, whose manifestations and duration depend on the virulence of S. sonnei strains under study. The modulating effect of S. sonnei on B-cell-mediated immune reactions is manifested by the inhibiting action of S. sonnei virulent strain and the stimulating action of S. sonnei vaccine strain on the formation of antibody-producing cells synthesizing S. sonnei lipopolysaccharide antibodies shortly after the injection of shigellae. The results of this study indicate that S. sonnei virulent and avirulent (vaccine) produce multifunctional and differing effects on cell-mediated immune reactions, these processes being dependent on the virulence of shigellae and their individual specific antigens.     

Specificity of the protective action of a ribosomal Shigella vaccine and the absence of activity in the ribosomes from R mutants

The ribosomal preparations of S. sonnei and some other bacterial species were obtained by the method of differential centrifugation, and the specificity of their protective action was studied in the keratoconjunctivitis test on guinea pigs. The ribosomal preparations were introduced parenterally in a single injection, and their protective action was determined two weeks later by the challenge of the animals with S. sonnei virulent strain and the subsequent calculation of the efficiency index (EI) by the formula: EI = C-V/C X 100, where C and V are the percentage of resistant eyes in the control and vaccinated groups of the animals respectively. For the ribosomal preparation obtained from a homologous avirulent strain this index was equal to 58%, while for the heterologous ribosomes obtained from Escherichia coli, Salmonella minnesota and S. flexneri in was close to zero. The ribosomal preparations obtained from S. sonnei R-strain which had no surface or cytoplasmic O-antigen also proved to be ineffective in rendering protection against local Shigella infection. The results of this investigation are compared with the data obtained by other authors, and the analysis of these results leads to the conclusion that the O-specific component is the indispensable factor of the protective activity of many ribosomal vaccines and its molecular properties require further study. The possible role of other components of the ribosomal vaccine is also discussed.     

The effect of a ribosomal Shigella sonnei vaccine on cellular immune reactions]

The influence of S. sonnei ribosomal vaccine on hematopoiesis, T- and B-cell-mediated immune reactions has been studied in the course of the development of experimental vaccinal process. The vaccine stimulated hematopoiesis, that was characterized by a dose-dependent increase in colony-forming units in the spleen (CFUs), a rise in CFUs in the blood and bone marrow and an increase in the pool of proliferating stem cells in bone marrow, shortly after injection. A pronounced immunostimulating effect of the vaccine on the formation of antibody-producing cells (APC) to heterologous antigen (sheep red blood cells) in the spleen has been established, and the vaccine has also been found to stimulate, though to a lesser extent, APC synthetizing specific antibodies to S. sonnei LPS. The injection of S. sonnei ribosomal vaccine influences the functional activity of effector T-cells; in its turn this phenomenon produces phasic changes in the migration activity of spleen cells in the presence of specific LPS and surface polysaccharide antigen of S. sonnei in phase I.     

Mechanism of the nonspecific protective action of Shigella antigens

The mechanism promoting the nonspecific action of antigens obtained from S. flexneri and S. sonnei by a sparing method has been studied. These antigens stimulate the T- and B- systems of immunity, that is followed by activation of myelopoiesis and the humoral protective factors of the body, which seems to underlie the formation of resistance to infection caused by nonspecific microorganisms.     

Stimulation of the secretory IgA system in monkeys by parenteral immunization with a ribosomal Sonne dysentery vaccine

The experiment was made on 16 monkeys (rhesus macaques). Only 1 out of 12 monkeys immunized with S. sonnei ribosomal vaccine and all 4 control monkeys fell ill as the result of oral challenge with S. sonnei virulent strain. The immunized monkeys stopped excreting Shigellae earlier than the control monkeys. Antibody to lipopolysaccharide (LPS) in the serum and saliva of the monkeys were studied in the enzyme immunoassay with monospecific antibodies to human IgA, IgG and IgM. A single injection of the ribosomal vaccine in a dose of 600 micrograms was shown to lead to a considerable increase in the levels of IgA, IgG and IgM antibodies to LPS in saliva. In parenteral immunization with the ribosomal vaccine the stimulation of secretory IgA system is similar to that resulting from oral challenge with Shigella virulent strain introduced in a dose of 50 X 10(9) microbial cells. No difference in the response of monkeys to primary and booster immunization was noted.     

Diagnostic test system for the quantitative determination of Shigella antigens in patient blood by an immunoenzyme analytical method

The diagnostic test system under trial has been shown to permit the detection of S. sonnei and S. flexneri specific antigens with an accuracy of 10(-3) micrograms. Along with high sensitivity, the test system has sufficiently high specificity. Statistically significant differences in the occurrence of specific dysentery antigens and their levels in the blood of dysentery patients and healthy persons have been revealed.     

A novel marker for the species-specific detection and quantitation of Shigella sonnei by targeting a methylase gene

Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qPCR primer set for the accurate diagnosis of Shigella sonnei was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.     

The structure of the population morbidity with specific intestinal infections in areas with different intensities in the development of the epidemic process and an evaluation of its manifestations]

The authors analyze the morbidity structure in five enteric infections (typhoid fever, dysentery caused by Shigella flexneri and Shigella sonnei, hepatitis A, and hepatitis E (non A, non B) with the fecal/oral mechanism of the agent transmission) in three towns of Turkmenia and in the town of Novomoskovsk, Tula Province. The incidence of S. sonnei dysentery was found higher in Novomoskovsk and that of the rest enteric infections under study in Turkmenia. The incidence of typhoid fever and hepatitis E was the highest among schoolchildren and adults, whereas preschool children suffered mostly from hepatitis A and S. sonnei dysentery. The authors discuss the specific features of the epidemic process manifestation in enteric infections.     

Latex test used for diagnosis of dysentery caused by Shigella sonnei]

The aim of this study was to evaluate the usefulness of latex reagent coated with immunoglobulins specific for antigens of phase I and II of S. sonnei for detection of these antigens in primary, mixed bacterial cultures. The study was performed on 919 fecal samples from individuals with clinical symptoms of dysentery, convalescents and from contact individuals. Material used for the test was bacterial suspension collected from McConkey or SS agars and a culture from selenite F broth heated at 100C. The results of the latex test were compared with the results of isolation of S. sonnei from the same cultures. S. sonnei was isolated from feces of 140 individuals (15.2%), while the latex test was positive in 215 cases (23.4%). The highest testing effectiveness , significantly higher than when isolation of pathogen was performed, was obtained only when 18-20 hr culture on Selenite F medium was used for latex test. The correlation between efficacy of testing for S. sonnei and phosphate content of Selenite F and a mode of its preparation was found. The latex test allows to eliminate from further bacteriological studies cultures free of S. sonnei thus it gives measurable economical profits and it shortens significantly time period of bacteriological examination.     

Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

A trivalent live Shigella vaccine candidate FSD01 against S. flexneri 2a, S. sonnei and S. dysen-teriae I was constructed. This candidate strain was based on the S. flexneri 2a vaccine T32. By homologous recombi-nation exchange, the chromosomal asd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while another asd gene of S. mutans was employed to construct an Asd complementary vector. This combination of asd 'host/ Asd vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S. sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the     

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7? distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.     

Acanthamoeba: could it be an environmental host of Shigella?

Shigellosis is a serious public health problem in Korea, because large outbreaks of Shigella sonnei infections were recorded in many parts of the country during the period 1998-2000. However, the epidemiological features of shigellosis are not well known. In this study, we devised conditions suitable for the growth and replication of Shigella in an amoebic intracellular environment, and investigate whether medium conditions affect the survival and replication of Shigella within Acanthamoeba. We evaluated the uptake rates of invasive and non invasive S. sonnei strains by three Acanthamoeba species, namely, A. castellanii Neff, A. astronyxis Ray & Hayes, and A. healyi OC-3A. When A. castellanii Neff was infected with S. sonnei 99OBS1 or 80DH248, shigellae was maintained for a longer time in cytoplasms than in other Acanthamoeba species. S. sonnei 99OBS1 strain (a virulent strain) was recovered in higher numbers than the non-virulent S. sonnei 80DH248 strain in all experiments. Moreover, S. sonnei was more easily engulfed by Acanthamoeba at 18 degrees C. The shigellae uptake rates of Neff strain, which was cultured in free-media (less nutrition), were higher (>10-fold) than those observed in original amoeba culture media (PYG medium) in all time points. S. sonnei 99OBS1 was localized, with an intact membrane, to the vacuoles of Acanthamoeba. We conclude that free-living amoebae more likely act as environmental hosts for shigellae, and thus, may have contributed to outbreaks of shigellosis in Korea.     

Ribosomal dysentery vaccine. II. Biological trials of active protection for guinea pigs and mice]

Ribosomal vaccine from Sh. sonnei injected subcutaneously once or twice in physiological saline or in Freund's complete adjuvant produces a marked protective effect against experimental keratoconjunctivitis in guinea pigs. Inhibition of the protective effect by high doses (above 100 microgram) of ribosomal vaccine is evident after a single, but not repeated injections. Protective effect in mice is achieved by immunization with very low doses of ribosomal vaccine: ED50 is 1.2 ng after challenge with 5.6 LD50. The nature of immunogenic factor responsible for the biological activity of the ribosome vaccine is still obscure. In contrast to Boivin's antigen, ribosomal preparations, even in high doses (1000--2000 microgram), have no toxic effect on mice and guinea pigs.     

Colicin Typing as an Epidemiological Tool in the Investigation of Outbreaks of Shigella sonnei

Shigella sonnei has become the most frequently reported cause of shigellosis in the United States. Since Shigella subgroup D has no other serotypes, colicin production has been used as a basis for differentiating and identifying epidemiologically related strains. The results of colicin typing 115 cultures of S. sonnei from eight outbreaks of shigellosis occurring in widely separated regions of the United States support the usefulness of this technique. In each outbreak, the cultures were either of the same colicin type or were uniformly untypable. Unrelated cases yielded a variety of types. Definitions of the relative frequencies and geographic distributions of the various strains of S. sonnei in the United States await an accumulation of experience with the method.     

综述与专论: 针对严重急性呼吸综合征冠状病毒2变异株Omicron的疫苗研究进展

2021年底,严重急性呼吸综合征冠状病毒2 Omicron变异株迅速取代Delta突变株在世界范围内广泛流行,其S蛋白具有36个位点突变,导致致病力和传播力发生明显变化,并且具备了免疫逃逸的能力。疫苗接种是目前疫情防控最普适的手段,研究发现,现有疫苗针对Omicron突变株的保护效果明显下降。新的免疫策略或特异性疫苗/多价疫苗针对Omicron有效性的评估均需要动物模型的支撑。在实验室条件下,利用动物模型进行活病毒攻击实验,是在体内验证保护性中和抗体、疫苗有效性的关键技术手段,本文将从动物模型方向综述国内外针对Omicron变异株的疫苗研究进展。     

Plasmid profile types of virulent and avirulent strains of phase-I Shigella sonnei and their rough phase-II and R-form variants

The study of S. sonnei in phase I, irrespective of their virulence, has revealed the existence of at least 3 types of profiles of large plasmids: (I)A having a single plasmid with a molecular weight of about 120 MD; (I)B having, alongside plasmid pSS120, a plasmid with a molecular weight of about 60 MD; (I)C, represented only by vaccine strain 6S, having three plasmids with molecular weights of about 80, 60 and 37 MD. The plasmid profiles of rough S. sonnei in phase II are characterized by the absence of large plasmids with a molecular weight of 120-80 MD, typical of bacteria in phase I, and can be in their turn subdivided, in accordance with the type of the initial culture, into three subvariants (II)A, (II)B and (II)C. The plasmid profiles of rough S. sonnei (R-forms and phase II) completely coincide. The biosynthesis of the specific antigen of S. sonnei in phase I can be determined by smaller derivatives obtained from large plasmid pSS120 by deletion (e.g., by a plasmid with a molecular weight of about 80 MD, such as plasmid pSS80).